The dopamine transporter (DAT) is the major molecular target responsible for both the rewarding properties and abuse potential of cocaine and related psychostimulants. The homologous neurotransmitter transporters (NTs) for serotonin and norepinephrine, SERT and NET, are primary targets of antidepressant drugs. These integral membrane proteins couple the accumulation of neurotransmitter to the movement of sodium ions down their concentration gradient. Progress in the study of their molecular structure and transport mechanisms has been hampered by an inability to develop high-level expression systems for these proteins and the subsequent lack of sufficient functional, purified protein. Bacterial membrane proteins are generally more amenable to structural analysis and high-level expression than are their eukaryotic counterparts. We have recently identified an entire family of proteins in archaea and in bacteria (currently 73 proteins from 45 different organisms) that are homologous to DAT. The sequence identity to DAT for the most similar proteins is approximately 25 percent, making it very likely that they have a similar structure. Our strategy was to develop a high-level expression system with one or more of these proteins to obtain adequate amounts for direct structural studies. During the first 1.5 years of our Stage I Cutting-edge Basic Research Award (CEBRA), we have: a) cloned 17 of these genes from various bacterial and archaeal genomes, b) heterologously over-expressed 12 of these in the membrane of E. coil, c) shown that one of these gene products, TnaT, is a sodium-dependent tryptophan transporter, confirming that these genes encode proteins with functions similar to the NTs, and reaffirming their value as models for direct structural analysis, d) purified full-length TnaT from the membrane to near homogeneity in yields of approximately 0.6 mg/I culture, e) constructed a cysteine-less TnaT that is functional and expresses at near wild-type levels, and f) constructed strategically placed individual cysteine mutants that express and function normally. In this Stage II CEBRA proposal the specific aims are: 1) To identify residues within or very near the substrate binding site in TnaT, a sodium-dependent tryptophan transporter from Symbiobacterium thermophilum, using mass spectroscopic analysis of azido-tryptophan analogs photo-incorporated into TnaT. 2) To identify a drug-like inhibitor of TnaT by screening a combinatorial chemical library. 3) To identify solubilization conditions that preserve the structure and function of TnaT. 4) To establish conditions for functional reconstitution of TnaT into proteoliposomes. When these aims have been achieved, we will be in a position to choose a limited number of the bacterial transporters for use in crystallization trials as a step towards obtaining a high-resolution structure. Moreover, we will also be poised to pursue spectroscopic methods to dynamic structure. Success in either or both of these goals would revolutionize our structural understanding of the function of related human neurotransmitter transporters in away that is only a remote prospect through continued work on the eukaryotic transporters alone.